Reduction of interference of immunoassays by substances derived from the framework regions of antibodies

ABSTRACT

The invention concerns an immunological process for the detection of an analyte in a sample, in particular of tumor markers wherein for the reduction of interference substances containing a peptide sequence derived from the framework regions of the variable domain of the antibodies to be detected or the antibodies used for immune therapy or scintigraphy are added to the test preparation. Furthermore, the invention concerns the use of such substances for the reduction of interference of immunoassays, a suppressive agent and a process for the reduction of interference of immunoassays by the substances mentioned.

The present invention concerns an immunological process for thedetection of an analyte in a sample, in particular of tumor markers,wherein substances containing a peptide sequence derived from theframework regions of the variable domain of the antibodies to bedetected or the antibodies used for immune therapy or scintigraphy isadded to the test preparation. The invention also concerns the use ofsuch substances for the reduction of interference of immunoassays, asuppressive agent and a process for the reduction of interference ofimmunoassays by the substances mentioned.

In the field of diagnostics especially immunological detection processeshave become very important during the last years. By these processesanalytes can be detected in biological samples. These analytes are forexample medicinal drugs, hormones, proteins, infectious agents,microorganisms and antibodies directed against these analytes. Inparticular in the diagnostics of cancer diseases the tumor antigens ortumor markers like e.g. CEA (carcinoembryonic antigen), PSA (prostatespecific antigen) or CA 125 are detected immunologically depending onthe disease.

All immunological detection reactions include a specific bindingreaction between the substance to be detected (analyte) and at least onespecific binding partner which specifically reacts with the analyte orwhich specifically binds to it. The analyte and the specific bindingpartner form a specific binding pair which generally is a complexbetween an antigen and an antibody or an antibody fragment. More thanone analyte or more than one binding partner can react with each otherin each reaction.

There are different possibilities how to detect these specific bindingreactions. In general one of the binding partners of the specificbinding reaction is labeled. Usual labelings are chromogens,fluorophores, substances capable of chemi- or electrochemiluminescence,radioisotopes, haptens, enzyme labels or substances which can formanother specific binding pair such as biotin/streptavidin.

An essential problem of immunoassays is related to the possibleunspecific binding reactions and undesired interactions between thespecific binding partners of the immunoassay and the sample components.Such interactions generally lead to an increase in the background signaland to a stronger signal variance and consequently to a reducedsensitivity and specificity of the corresponding test. Depending on thekind of interference provoked by unspecific interactions false positiveor false negative test results can occur.

This is the case when interference factors occur in human sera andparticularly interact with immunoglobulin reagents of an immunoassayfrequently used as binding partners and may thus have an effect on theimmunoassay. Especially in immunoassays with two monoclonal antibodiesconsiderable measuring errors with serious consequences for the furthertreatment of a patient (Kinders and Hass (1990) Eur. J. Cancer 26 (5),647-648; Boscato and Stuart (1988) Clin. Chem. 34 (1), 27-33) may occur.

A large number of these interference factors can be classified as HAMA(human anti-mouse antibodies) which are antibodies present in the sampleto be tested and directed against the specific antibodies used asreagents. Often the name HAMA-interference is also used for antibodyinterferences which were not provoked by contact with mouseimmunoglobulin or which were not strictly specific for mouseimmunoglobulin. By this HAMA interference unspecific cross-linking ofthe antibody bound to the solid phase with the labeled antibody to bedetected can for example occur in a conventional sandwich assay despitethe absence of the analyte. As a consequence, a false positive signalresults.

This problem has become bigger since recently immunoscintigraphy hasbeen used more and more frequently in the diagnostics and therapy oftumors. For this purpose radioactively labeled monoclonal antibodies areinjected in the bloodstream of the patient. Then the labeled antibodiesspecifically bind to the relevant tumor tissue. By subsequent scanningof the radioactivity, e.g. with a scintillation camera the tumor can belocalized exactly. Immunologically stimulating therapies—even withmonoclonal antibodies—are also used frequently to stimulate theformation of tumor-specific antibodies for the control of the tumor inthe patient's organism.

When using these processes very high and partly very specific HAMAtiters occur, as mentioned above, with increasing frequency in thepatient sera (see for example Kath. et al. (1996) Oncology 2, 287-296;Holz et al. (1996) Clin. Immunther. 5 (3), 214-222; White et al. (1997)Europ. J. of Cancer 33(5), 40; Baum et al. (1993) Hybridoma 12(5),583-589; Donnerstag et al. (1995) Int. J. Oncology 6, 853-858;Livingston et al. (1995) Vaccine Research 4 (2), 87-94; Jeweid et al.(1996) Cancer 78 (1), 157-168). They can also provoke interferences intests involving chimeric antibodies. An important example of antibodiesapproved in the tumor therapy of colon carcinoma in Germany is theantibody 17-1A commercially named Panorex (Kath et al. (1996), supra;Holz et al. (1996), supra). In addition the list of the antibodies usedin clinical studies includes a large number of further therapeuticreagents containing antibodies which very probably will be approved.Antibodies against CEA, CA125 and CA72.4 are already clinically appliedin the immunological scintigraphy and it is expected that they will beused with increasing frequency too.

For state of the art elimination of these interference factors, oftenunspecific immunoglobulins or fragments thereof are used which arederived from the same strain of animal species as the antibodies used asreagents in the test. Thus alternative binding sites are offered to theinterference factors where they can be taken up so that the specificimmune reaction between the analyte and the antibodies is no moredisturbed. For elimination of unspecific reactions in immunoassays usingmonoclonal mouse antibodies as reagents the addition of mouse or ratserum and, respectively, ascites is described in EP-A-0 174 026. Afurther possibility to avoid such interference is the addition ofpurified monoclonal Fab fragments or IgG molecules derived from themouse which especially in a polymerized form develop a good suppressiveeffect according to U.S. Pat. No. 4,914,040.

To avoid false positive reactions for the detection of CEA the additionof a mixture of IgG molecules of different classes (IgGI, IgG2a, IgG2b)is recommended in the WO 91/16627. The provision of differentimmunoglobulin preparations in the quantities known and of a constantquality requires, however a lot of experimental time and mainly concernsthe constant regions of the antibodies.

The reduction of the interference potential in the constant part of theantibody can also be achieved by the use of Fab fragments instead ofintact immunoglobulins since the mainly occurring Fc interferences donot get a chance here due to the lacking Fc part. An optimized versionare humanized antibodies with an Fv region composed of one mouse and onehuman part so that the susceptibility to interference can be furtherreduced even in the variable part (Kuroki et al. (1995) J. of Immunolog.Methods 180, 81-91). The use of chimeric antibodies with a variablemouse part and human constant regions can also reduce the susceptibilityto interference of diagnostic tests.

According to the state of the art the addition of a protein and inparticular of antibodies immunologically related to the antibodies usedas detection reagents to avoid unspecifically increased or decreasedvalues in the detection of an analyte is described too (EP-A-0 296 544).Due to the similarity of the antibodies added to the reagent antibodiesused the substance interfering with these antibodies is capturedselectively. For the provision of the suppressive antibodies it isrecommended in the EP-A-0 296 544 to cleave off the antigen-bindingstructures of the reagent antibodies used, to block the antigen-bindingstructure by a complementary antigen structure, to change the antigenstructure of the reagent antibody used by mutation or to useanti-idiotypical antibodies of the same subclass.

With the state of the art processes it is, however not possible toeliminate without much experimental input the interferences observed inthe samples of patients previously treated with immunotherapeuticmethods and which are very individual depending on the therapeuticantibody applied.

It was therefore an object to develop an improved process for thedetection of an analyte and in particular tumor markers which wouldavoid any interference by unspecific substances such as HAMA to a largeextent and better than with the state of the art. methods.

This object is achieved by a new immunological process for the detectionof an analyte in a sample wherein for the reduction of interferencesubstances are added to the test preparation containing a peptidesequence derived from the framework regions of the variable domain ofthe detection antibody or of the antibodies used for immunologicaltherapy or scintigraphy.

In particular, substances are used containing sequence and/or structurecharacteristics occurring in the Fv regions of at least one of thespecific detection antibodies used as a reagent in an immunoassay orwhich correspond to the sequences of a therapy/immunoscintigraphy whichavoid any interference by HAMA almost completely. A particularly goodsuppressive effect can be observed when the substances used containsequence or structure characteristics which occur in framework 1 orframework 3 of the heavy chain of the specific antibody/ies.

BRIEF DESCRIPTION OF THE DRAWINGS

For further explanation FIG. 1 lists the heavy chain sequences of thevariable region of various antibodies one below the other. It becomesclear that in the framework region 1 (amino acid position 1 to 30) thereis very much homology and to a large extent even identity among thetumor antibodies listed (OC125, B72.3, CD4 and PSA-M66). The same istrue for the framework region 3 (amino acid position 69-100). The degreeof homology in the heavy chain sequences of the variable region of thesingle tumor antibodies is here high too. A comparably low homologyexists however between the sequences of the tumor antibodies and therespective sequences of the framework regions 1 and 3 of the non-tumorantibodies MAB33 and TSH antibody (TSH=thyroid stimulating hormone). Theantibody B72.3 is described in xiang et al. 1990, Mol. Immunol. 27, p.809-817 as well as in J. Immunology (1993), 151, 6559-6568; the antibodyMAB33 is described in Buckel et al. 1987, Gene 51, 13-19; the antibodyTSH-A8 in the EP-A-0 378 175; the antibody OC125 in Tumor Biology(1996), 17, 196-219 and 325-331 as well as in Hybridoma (1994) 13(5),359-365).

According to the state of the art it has not been recognized that theimmunological relationship of the antibodies is to be attributed tosequence homologies in the variable region of the antibody. Especiallyfor HAMA interferences in patient samples showing very high and specificHAMA titers due to immunological scinitigraphy or immunologicallystimulating therapies also by tumor-antigen directed mab the solutionaccording to the invention is superior to that of the state of the artsince these treatments with monoclonal antibodies lead to special,non-idiotypical but among themselves similar HAMA interferences in thesamples to be tested particularly in different tumor marker tests.

The framework regions of an antibody are located in the variable part(Fv) of the molecule but do not really take part in antigen binding.They are essential for the characteristic folding of the variable partof the antibody and maintain the framework and structure of the antigenbinding site (see for example Roitt et al. (1989) Immunology, GowerMedical Publishing, London, N.Y., chapter 7, Antigen Recognition).

For the process according to the invention antibodies are preferablyused as the suppressive substance. The use of antibodies and theirfragments has been proven to be particularly appropriate; their aminoacid sequence of the framework region corresponds to the sequence of thedetection antibodies used as reagents or the antibodies needed fortherapy or immunological scintigraphy.

The monoclonal antibodies B72.3 (J. Immunology (1993), 151, 6559-6568)and OC125 (Tumor Biology (1996), 17, 196-219 and 325-331; Hybridoma(1994) 13 (5), 359-365) are used particularly preferably for thereduction of interference (see also the ISOBM report: Tumor Biology1996; 17:196-219). Antibodies showing structure characteristics orbinding properties equivalent to those of the antibodies B72.3 and OC125can also be used for the reduction of interference.

For the suppressive effect of the antibodies used the amino acidsequence and the folding structure in the framework region 1 or 3 areessential. It should correspond to the sequences listed in FIG. 1 orshould at least be very homologous to it. The homology is sufficientwhen a suppressive effect can be observed while using a suppressiveantibody. In principle all antibodies having—in the framework regions 1and/or 3 — one of the amino acid sequences listed in SEQ ID NO 1 to 8are suitable for reduction of interference. The origin and theproduction of the antibodies used for the reduction of interference canbe chosen at will. The monoclonal antibodies can be derived from ascitesmaterial or produced in the bioreactor according to methods known to theexpert. The suppressive antibodies can but do not have to be derivedfrom the same organism as the detection antibodies.

According to the invention HAMA interferences can be considerablyreduced especially in processes for the detection of tumor markers. Thereduction of interference of different tumor marker tests by thesuppressive agent according to the invention is therefore surprisingsince tumor antigens are quite different in their amino acid sequencesand structure. Surprisingly, it is however possible to suppressinterferences in different tumor marker tests with onesequence-homologous reagent. One subject matter of the invention istherefore an immunological process for the detection of a tumor markerwith reduction of interference by adding to the test preparation atleast one substance—preferably antibodies—containing a peptide sequencederived from the framework regions of the variable domain of theantibodies to be detected or the antibodies used for immune therapy orscintigraphy.

The detection of the tumor markers CA125, CA15-3, CA19-9, CEA, PSA (in afree and complexed form) and AFP belong to the tests suppressedpreferably. According to the invention processes for the detection ofanalytes not associated to tumor diseases, such as cardiovascularmarkers (like troponin T or myoglobin) are however very well suppressedtoo.

The term antibody means polyclonal or monoclonal antibodies. It alsomeans the whole antibody as well as all fragments thereof usuallyapplied in immunoassays and other uses, such as F(ab)′₂ Fab′ or Fabfragments. The antibodies are produced according to methods known to theexpert and can also be antibodies produced for example by geneticengineering. It is important that all substances used for reduction ofinterference contain one peptide sequence derived from the frameworkregions of the variable domain Fv of the detection antibodies.

The term detection antibody refers to immunoglobulins and theirconjugates which are used as detection reagents in the test. In the caseof a conventional sandwich assay the antibody bound to the solid phaseand specifically binding to the analyte as well as the detectionantibody specifically binding to the analyte on the one hand andcarrying a label on the other hand belong to the detection antibodies.If further antibodies are necessary for the label detection (forinstance in the case of the digoxigenin label which is detected with anenzyme labeled anti-digoxigenin antibody) they are also referred to asdetection antibodies.

According to the invention one or several fragments of the frameworkregions—especially of the framework regions 1 and 3 of the Fv domain ofthe heavy immunoglobulin chain—can also be used as suppressivesubstances. These fragments are preferably peptides which can besynthesized by enzymatic digestion of the intact antibodies or bychemical reactions according to methods known to the expert.

Peptides containing peptide sequences from the framework regions 1 and 3of the MAB OC125, B72.3, PSA or CD4 are suitable as substances used forthe reduction of interference of immunological processes.

From the framework region 1: QVQLQQSGPEASVKMSCKASGYIFT (SEQ ID NO 1 fromMAB OC125) QVQLQQSDAEASVKISCKASGYTFT (SEQ ID NO 2 from MAB B72.3)QVQLQQSGAEASVKISCKATGYTFS (SEQ ID NO 3 from MAB <PSA>M66)QVHLQQSGPEPSVKMSCKASGYTFT (SEQ ID NO 4 from MAB <CD4>)

From the framework-region 3: RATLTVDKSSSTAYMQLNSLT (SEQ ID NO 5 from MABOC125) KATLTADKSSSTAYMQLNSLT (SEQ ID NO 6 from MAB B72.3)RATFTADSSSNTAFMEFGSLT (SEQ ID NO 7 from MAB<PSA>M66)KAILTADKSSSTAYMEFSSLT (SEQ ID NO 8 from MAB<CD4>)

Partial sequences of the peptides mentioned before with a length of atleast 6 amino acids are also appropriate for the reduction ofinterference. The peptide sequences do not have to be present in anisolated form. They can also be flanked by further amino acid sequenceswhich are not derived from the variable part of the antibody. It is alsopossible that the peptides are flanked by carbohydrate or lipidresidues. A modification condition is however that the suppressiveeffect is maintained and that the modifications themselves do notprovoke interferences within the immunoassay.

It is also conceivable that the peptides used for reduction ofinterference carry the corresponding sequence even several times, i.e.in a multimer form, and can thus also be used as polyhaptens. This meansthat for the production of a polyhapten the peptides according to theinvention are coupled several times to one carrier. Different peptidesaccording to the invention can be coupled to one carrier too. Amacromolecule which does not take part in the immunological reactionitself, e.g. a bigger protein such as bovine serum albumin, latexparticles, polystyrene, gold or dextran can serve as a carrier. Theproduction of polyhaptens can be analogous to the method described in WO96/03652.

The advantage of the substances for reduction of interference accordingto the invention is that for the removal of HAMA interferences a time-and cost intensive pretreatment of the samples such as PEGprecipitation, protein A/G chromatography or heat treatment can beomitted.

According to the invention the test procedures and test formats can—inprinciple—be chosen arbitrarily. Homogeneous and heterogeneousprocedures known to the expert are conceivable. The test formatspreferred are heterogeneous formats with one specific binding partnercoupled directly or indirectly to a solid phase. The conventionalantigen detection in a sandwich format which is preferably used in tumormarker tests is mentioned here as an example. In the sandwich format theanalyte (in our case an antigen) is bound like a sandwich between anantibody bound to a solid phase and a labeled antibody. The labeling isdetected in one of the phases after the separation of the solid phasefrom the liquid phase.

Another example of the heterogeneous test procedure is the indirectdetection of an analyte antibody via its binding to a solid-phase boundantigen. Here, the antibody is detected by a further labeled antibody'sbinding to the analyte antibody.

A further test format which can be suppressed according to the inventionis the competitive test procedure with one solid-phase bound complexformed by two binding partners or antibodies specific for each otherwhere the binding partner not directly bound to the solid phase islabeled. The analyte which depending on the test requirements is anantigen or an antibody displaces the labeled binding partner dependingon its concentration from the complex. After separating the solid phasefrom the liquid phase the label is detected in one of the phases. Alsoin this case the suppressive substances according to the invention blockthe unspecific binding of interfering sample substances to the bindingpartners/antibodies used as detection reagents and thus avoid false testresults as far as possible.

The maximum concentrations of the antibodies in the sample preparationused for reduction of interference are only given by the solubility ofthe antibodies in aqueous medium. Minimum concentrations ofapproximately 10 μg/ml to 40 μg/ml and upper limits of approximately 2mg/ml, preferably 250 μg/ml antibody have proven to be appropriate.

Besides the substances according to the invention further suppressivemeasures can be taken if this is required. One of these measures is forinstance the addition of MAB33 (Boehringer Mannheim GmbH, Germany,Ident. No. 1200 941) or of other antibodies with the same effect, polyMAB 33 (polymer mouse IgG, Boehringer Mannheim GmbH, Germany, Ident. No.1368 338), RSA, salts, detergents.

Besides the so-called wet tests with test reagents in a liquid phase allother usual dry test formats appropriate for immunological detection ofanalytes can be used. In these dry tests or test strips as they are forexample described in EP-A-9 186 799 the test components are applied onone carrier. In this case the suppressive agent according to theinvention is applied to the dry test strip before the immunologicalreaction.

Preferably, the suppressive agent according to the invention should beadded to the sample before or simultaneously with the binding partnersused as detection reagents to enable as early as possible a reaction ofthe interfering unspecific substances with the suppressive agent.

All usual biological liquids known to the expert can be used as samplesfor the procedure of the immunoassays to be suppressed according to theinvention. The substances preferred as samples are body liquids such aswhole blood, blood serum, blood plasma, urine or saliva.

A further subject matter of the invention is a suppressive agentcontaining at least one substance including a peptide sequence derivedfrom the framework regions of the variable domain of the antibodies tobe detected or the antibodies used for immune therapy or scintigraphy.Further components of the suppressive agent can be buffers, salts anddetergents known to the expert. The suppressive reagent can be providedin a liquid, aqueous or in a lyophilized form.

A further subject matter of the invention is a process for reduction ofinterference of immunoassays wherein especially for the suppression ofHAMA effects substances containing a peptide sequence derived from theframework regions of the variable domain of the antibodies to bedetected or the antibodies used for immune therapy or scintigraphy areadded to the test preparation.

The invention is further described by the following examples.

EXAMPLE 1 Reduction of Interference of the CA125 Test with DifferentSuppressive Reagents

The CA 125 detection is performed according to the procedure describedin the package leaflet of the Enzymun-Test® CA 125 II of BoehringerMannheim GmbH, Germany (ident. No. 1 289 004). The test principle is a1-step-sandwich ELISA based on the streptavidine technology. Twomonoclonal CA125-specific antibodies specifically recognizing differentepitopes on the CA125 are used. One of the antibodies is labeled withbiotin and the other antibody with peroxidase. Together with anincubation solution containing biotinylated anti-CA125 antibodies andanti-CA125 antibodies labeled with peroxidase (POD) the sample is filledinto a plastic tube coated with streptavidine. During the incubation thebiotinylated antibody binds to the solid phase. The CA125 present in thesample binds to the biotinylated antibody. The antibody labeled with PODbinds to the CA125 which is bound to the biotinylated antibody.

The sandwich complexes bound to the solid phase are detected by anindicator reaction after a washing step. For this, a substrate-chromogensolution containing ABTS®(2,2′-azino-di-[3-ethyl-benzthiazoline-sulfonic acid (6)]-diammoniumsalt) and H₂O₂ is added to the preparation. By the enzymatic activity ofthe peroxidase and depending on the amount of bound POD-labeled antibodya dye is built which can be detected photometrically.

Together with the biotinylated antibody 40 μg/ml of differentsuppressive state of the art reagents and—for comparison reasons—thesuppressive reagent according to the invention are added to the testpreparations.

The following substances are used as suppressive reagents: the solutionaccording to the invention on the basis of the example MAB B72.3 (intactIgG), a HAMA suppressive reagent of the company Trina (mouse IgG), aHAMA suppressive reagent of the company Scantibodies (HBR, monoclonalanti-human IgM), the suppressive reagent MAB33 of Boehringer MannheimGmbH, Germany (ident. No. 1 200 941), the suppressive reagent pMAB33 ofBoehringer Mannheim GmbH, Germany (ident. No. 1 096 478) as well asthree tumor marker antibodies against PSA, AFP and CEA.

Serum samples of patients with an ovary carcinoma having undergone animmune scintigraphy as well as commercially available HAMA panels andone normal serum (NS) of a healthy patient are used as samples. Table 1shows the measuring values of the CA125 test with different suppressivereagents added. One POD unit (I U) corresponds to the enymatic activityoxidizing 1 μnol ABTS in one minute at 25° C. and a pH of 5.

TABLE 1 Values measured with the CA125 test CA125 CA125 + WithoutCA125 + Trina- CA125 + CA125 + CA125 + CA125 + CA125 + CA125 + additionB72.3 13 HBR MAB33 pMAB33 <PSA>−M66 <AFT>MTu11 <CEA>MTu3 U/ml U/ml U/mlU/ml U/ml U/ml U/ml U/ml U/ml Normal serum 16.42 23.4 20.64 15.48 14.6215.12 9.25 12.5 7.16 Immune scinti- >496 107.4 124.68 143.05 434.29433.36 221.04 350.42 369.78 graphy with B43.13 (Ovary carcinoma) Immunescinti- 129 118.6 117.54 117.13 134.43 126.6 125.8 141.98 130.1 graphywith B43.13 (Ovary carzinoma) Immune scinti- 262 96.5 106.65 117.31158.04 160.89 111.9 157.97 158.63 graphy with B43.13 (Ovary carcinoma)Commercially 111.7 — 34.42 35.3 50.77 55.65 — — — available HAMA- PanelIII (Bioclinical Partners) Immune >496 289.7 329.93 322.77 348.52 360.41300.2 402.03 335.33 scintigraphy with OC125 (Ovary carcinoma)Commercially 382.2 22.2 115.05 137.75 95.6 111.84 114.6 129.03 120.02available HAMA- Panel (Starrate D) Immune >496 158 404.09420.97 >496 >496 — — — scintigraphy with B43.13 (Ovary carcinoma)

Without suppressive reagents the CA125 test indicates positive measuringvalues which are however false positive values. It can be shown thatwith the monoclonal antibody B72.3 the best HAMA suppression. i.e. thelowest measuring value can be achieved. The suppressive agent accordingto the invention is thus clearly better than those of the state of theart. Among the tumor marker antibodies the PSA antibody has proven to bethe most appropriate one for reduction of interference what is analogousto the sequence homology.

EXAMPLE 2 Reduction of Interference of the CA125 Test with <CD4>M3-10.MAB33 and the Panorex Antibody

The detection of CA125 is performed according to example 1. Forcomparison the suppressive reagent according to the invention (here: MABB72.3). polymerized and non-polymerized MAB33 as in example 1. theantibody <CD4>M3-10 and the Panorex antibody are used as suppressivereagents. The suppressive reagents are added in a concentration of 40μg/ml.

TABLE 2 Values measured with the CA125 test CA125 without CA125 +CA125 + CA125 + CA125 + CA125 + addition B72.3 Panorex <CD4> PMAB33MAB33 U/ml U/ml R % U/ml R % U/ml R % U/ml R % U/ml R % TMC1 43.05 42.8899 44.73 104 38.98 90 47.97 111 40.41 94 TMC2 88.68 90.13 102 94.37 10685.45 96 92.77 105 87.53 99 Normal serum 21.13 19.43 92 21.99 104 15.2272 23.19 110 21.89 104 Commercially 60.83 39.47 65 50.8 84 40.41 66 48.380 42.58 70 available HAMA- Panel I (Bioclinical Partners) Immune 142.9477.44 54 93.98 66 77.61 54 89.87 63 77.82 54 scintigraphy with B43.13(Ovary carcinoma) Commercially 408.08 38.62 9 57.06 14 42.63 10 57.16 1467.83 17 available HAMA- Panel (StarrateD) Conmmercially >519 69.2 <13513.82 <99 77.78 <15 111.72 <21 147.35 28 available HAMA- Panel II(Bioclinical Partners) Immune >519 358.66 <70 1350.1 — 465.88 90 >519 —1189.08 — scintigraphy with OC125 (Ovary carcinoma) Immune 239.26 39.6417 119.11 50 45.84 19 103.22 43 112.67 47 scintigraphy with B43.13(Ovary carcinoma) Immune 211.88 43.73 21 71.92 34 62.07 29 83.91 40108.34 51 scintigraphy with B43.13 (Ovary carcinoma) %R: percentage ofrecovery amount received in relation to the test without addition ofsuppressive reagent TMC1 and TMC2: Tumor marker control of BoehringerMannheim GmbH, Germany (ident.-No. 1 489 666), control serum on thebasis of human serum with two required values, containing among othersAFP, CEA, PSA, free PSA, CA125

It is shown that with the sequence homologous <CD4> antibody thesuppression achieved is comparable to that of the antibody B72.3 butthat the less sequence homologous Panorex antibody has a considerablylower suppressive effect (only a small signal decrease). In addition,the considerably lower effect of the state of the art suppressivereagents becomes evident.

EXAMPLE 3 Reduction of Interference of the CEA Test with DifferentSuppressive Reagents

To get a second criterion of the suppressive effect besides the CA125test the CEA-Enzymun® test of Boehringer Mannheim GmbH (ident. No.1448021) was chosen as matrix. The process is performed according to theprocedure described in the package leaflet. Like in the CA125 test thetest principle is also a 1-step-sandwich ELISA based on thebiotin-streptavidine technology. Two monoclonal CEA-specific antibodiesrecognizing different epitopes on the CEA are used. One of theantibodies is labeled with biotin and the other antibody withperoxidase. Together with an incubation solution containing biotinylatedanti-CEA antibodies and anti-CEA antibodies labeled with peroxidase(POD) the sample is filled into a plastic tube coated withstreptavidine. During the incubation the biotinylated antibody binds tothe solid phase. The CEA present in the sample binds to the biotinylatedantibody. The antibody labeled with POD binds to the CEA which is boundto the biotinylated antibody.

The detection of the sandwich complexes bound to the solid phase isperformed via the indicator reaction as described in the examples 1 and2.

Reagents mentioned in the examples 1 and 2 as well as the OC125 antibodyare used as suppressive reagents. They are added in a concentration of10 μg/ml.

TABLE 3 Values measured with the CEA test CEA CEA CEA CEA Enzymun +Enzymun + Enzymun + Enzymun + CEA Enzymun OC125 B72-3 HBR MAB33 ng/mlng/ml ng/ml ng/ml ng/ml Normal serum 0.79 1.01 — 1 1.1 Immunescintigraphy with 6.8 2.25 2.78 2.77 5.3 B43.13 (Ovary carcinoma) Immunescintigraphy with 7.84 3.01 3.62 3.4 6.09 B43.13 (Ovary carcinoma)Commercially available HAMA- >51.6 4.98 6.89 9.1 17.72 Panel III(Bioclinical Partners) Immune scintigraphy with 13.7 3.9 — 5.32 8.14B43.13 (Ovary carcinoma) Commercially avaiable HAMA- 5.77 2.94 2.8  2.13.95 Panel (Starrate D) Commercially available HAMA- 12.97 4.2 3.62 4.425.36 Panel II (Bioclinical Partners) Immune scintigraphy with 48.6210.45 8.28 8.67 19.8 <CEA> (Colon carcinoma)

The results obtained with the suppressive reagents according to theinvention MAB B72.3 and OC125 are to a certain extent comparable. Excepta few amino acids these two antibodies are sequence homologous in theframework regions 1 and 3. The state of the art results of thesuppressive effect are clearly worse.

EXAMPLE 4 Reduction of Interference of Different Test Systems

For comparison different tumor marker tests are shown that on the onehand are based on the Enzymun® test procedure (s. examples 1 to 3) andon the other hand on the Elecsys® procedure of Boehringer Mannheim GmbH.The Elecsys® procedure for all detections is based on the1-step-sandwich principle with two antibodies recognizing differentepitopes on the analytes (tumor markes) analogously to example 3. Thesolid phase is formed of magnetic latex beads coated with streptavidineto which binds a biotinylated antibody specific for the tumor marker.The bound tumor marker is detected after the separation of the solidphase from the liquid phase by measuring the electrochemiluminescenceresulting from a second tumor-marker specific antibody labeled with aruthenium complex.

Here, the suppressive effect of the solution according to the inventionwas tested with different Enzymun® and Elecsys®-tests:

CEA-Enzymun® (Ident.-No. 1448021; CEA Elecsys® (Ident.-No. 1731629);CA125 Enzymun® (Ident.-No. 1289004). CA125 Elecsys® (Ident.-No.1776223); AFP Elecsys® (Ident.-No. 1731327); PSA tot. Enzymun®(Ident.-No. 1555332); PSA tot. Elecsys® (Ident.-No. 1731262); PSA freeEnzymun® (Ident.-No. 1776444); PSA free Elecsys® (Ident.-No. 1820800).

The suppressive reagent MAB B72.3 is added in a concentration of 40μg/ml.

The samples are high-titer HAMA sera from immune therapy orscintigraphy.

TABLE 4 Reduction of interference of different test systems PSA PSA PSAPSA CA125 CA125 AFP PSA total PSA tot. PSA free PSA free CA125 Enz. +CA125 ECL + AFP ECL + total Enz. + tot. ECL + free Enz. + free ECL +Enz. B72.3 ECL B72.3 ECL B72.3 Enz. B72.3 ECL B72.3 Enz. B72.3 ECL B72.3Serum 1 339.1 24.9 21.6 — 16.98 12 1.62 0.2 0.73 0.2 0.35 0.12 0.73 0.15Serum 2 >496 52.7 72.71 46.6 6.36 2.81 0.48 0 1.21 0.36 1.04 0.28 2.60.21 Serum 3 >496 370.2 186.6 133.1 4.73 1.87 0.75 0 1.12 0.17 1.62 0.324.93 0.27 Serum 4 >496 82.3 59.6 30.1 16.07 5.9 1.3 0.13 1.7 0.52 2.840.39 13.1 0.44 Serum 5 205.8 23.01 24.3 2.33 1.85 0.07 0 0.27 0.05 0.110.03 2.23 0.12 Serum 6 181.1 28.65 29.62 2.03 1.52 0.07 0 0.38 0.1 0.250.08 3.57 0.24 Serum 7 — — — 2 1.24 0.14 0 0.41 0.14 0.45 0.18 2.7 0.34Enz. = Enzymun ® ECL = Elecsys ®

With CA125 both tests show a considerable reduction of the HAMAinterference by addition of B72.3.

The interfering potential is also decreased with AFP. PSA total andfree. With free PSA the interference susceptibility of the Elecsys® testis however higher than that of the Enzymun® test. A suppressive effectcan thus be shown regardless of the test system.

EXAMPLE 5 Reduction of Interference in the Troponin-T and MyoglobinTests

To show the suppressive effect also with other parameters than tumormarkers the cardiovascular markers troponin T (Ident. No. 1 731 3 51)and myoglobin (Ident. No. 1 820 788) are chosen.

The test principle is based on the Elecsys® test described in example 4.Two monoclonal antibodies against troponin T and, respectively,myoglobin recognizing different epitopes on the analyte (troponin T andmyoglobin) are used. One of the mab is labeled with biotin the otherwith a ruthenium complex the electrochemiluminescence signal of which ismeasured.

100 μg/ml of the suppressive reagent MAB B 72.3 are added to the testpreparations.

TABLE 5 Reduction of interference of tests for the detection of troponinT and myoglobin Myoglobin Myoglobin + 100 μg/ml B72.3 Troponin TTroponin T + 100 μg/ml B72.3 (ng/ml) (ng/ml) R % (ng/ml) (ng/ml) R %Control 1 99.229 100.396 101 0.148 0.148 100 Control 2 1312.549 1283.68898 7.261 7.325 101 Immune therapy with 19.819 14.888 75 0.015 0.011 73B43.13 (Ovary carcinoma) Commercially available 334.348 60.72 18 0.1190.073 61 HAMA-Panel (Starrate D) Commercially available 210.342 42.31820 0.187 0.121 65 HAMA-Panet (Bioclinical Partners) Immune therapy with231.522 46.914 20 0.178 0.106 60 OC125 (Ovary carcinoma) Immunescintigraphy - 56.877 21.099 37 0.056 0.026 46 <CEA>- MABs Immunetherapy with 31.062 19.405 62 0.061 0.031 51 B43.13 (Ovary carcinoma)

Here too, the addition of B72.3 leads to remarkably lower signals andcancels the false positive measurement. The application of thesuppressive reagents according to the invention is therefore also shownfor markers other than tumor markers.

14 1 25 PRT Mus musculus 1 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu AlaSer Val Lys Met Ser 1 5 10 15 Cys Lys Ala Ser Gly Tyr Ile Phe Thr 20 252 25 PRT Mus musculus 2 Gln Val Gln Leu Gln Gln Ser Asp Ala Glu Ala SerVal Lys Ile Ser 1 5 10 15 Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 3 25PRT Mus musculus 3 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Ala Ser ValLys Ile Ser 1 5 10 15 Cys Lys Ala Thr Gly Tyr Thr Phe Ser 20 25 4 25 PRTMus musculus 4 Gln Val His Leu Gln Gln Ser Gly Pro Glu Pro Ser Val LysMet Ser 1 5 10 15 Cys Lys Ala Ser Gly Tyr Thr Phe Thr 20 25 5 21 PRT Musmusculus 5 Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr MetGln 1 5 10 15 Leu Asn Ser Leu Thr 20 6 21 PRT Mus musculus 6 Lys Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu AsnSer Leu Thr 20 7 21 PRT Mus musculus 7 Arg Ala Thr Phe Thr Ala Asp SerSer Ser Asn Thr Ala Phe Met Glu 1 5 10 15 Phe Gly Ser Leu Thr 20 8 21PRT Mus musculus 8 Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr AlaTyr Met Glu 1 5 10 15 Phe Ser Ser Leu Thr 20 9 114 PRT Mus musculus 9Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 1015 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr Tyr 20 2530 Met Lys Trp Val Lys Gln Ser His Gly Arg Glu Trp Ile Gly Asp Ile 35 4045 Asn Leu Asn Asn Gly Asp Thr Phe Tyr Asn Gln Lys Phe Lys Gly Arg 50 5560 Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu 65 7075 80 Asn Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser 8590 95 Asp Asp Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val100 105 110 Ser Ser 10 114 PRT Mus musculus 10 Gln Val Gln Leu Gln GlnSer Asp Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile SerCys Lys Ala Ser Gly Tyr Thr Phe Thr Asp His 20 25 30 Ala Ile His Trp AlaLys Gln Lys Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser ProGly Asn Asp Asp Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala ThrLeu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu AsnSer Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Lys Arg Ser TyrTyr Gly His Trp Gly Gln Gly Thr Thr Val Thr Val 100 105 110 Ser Ser 11119 PRT Mus musculus 11 Gln Val His Leu Gln Gln Ser Gly Pro Glu Leu ValLys Pro Gly Pro 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly TyrThr Phe Thr Asp Tyr 20 25 30 Val Val Ser Trp Met Gln Gln Arg Thr Gly GlnVal Leu Glu Trp Ile 35 40 45 Gly Glu Ile Tyr Pro Gly Ser Gly Ser Ala TyrTyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Ile Leu Thr Ala Asp Lys SerSer Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Ser Ser Leu Thr Ser Glu AspSer Ala Val Phe Phe Cys 85 90 95 Ala Arg Arg Gly Asp Gly Ser Leu Gly PheAla His Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ala Ala 115 12117 PRT Mus musculus 12 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu ValLys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly TyrThr Phe Ser Ser Tyr 20 25 30 Trp Ile Glu Trp Met Lys Gln Arg Pro Gly HisGly Leu Glu Trp Ile 35 40 45 Gly Asp Phe Leu Pro Gly Ser Gly Ser Ser TyrPhe Asn Glu Lys Phe 50 55 60 Lys Gly Arg Ala Thr Phe Thr Ala Asp Ser SerSer Asn Thr Ala Phe 65 70 75 80 Met Glu Phe Gly Ser Leu Thr Ser Glu AspSer Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Ala Gly Arg Val Asp TyrTrp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ala 115 13 123 PRTMus musculus 13 Glu Val Gln Gly Val Glu Ser Gly Gly Gly Leu Val Lys ProGly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr PheSer Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg LeuGlu Trp Val 35 40 45 Ala Thr Ile Ser Asp Gly Gly Ser Tyr Thr Tyr Tyr ProAsp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys AsnAsn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr AlaMet Tyr Tyr Cys 85 90 95 Ala Arg Asp Lys Ala Tyr Tyr Gly Asn Tyr Gly AspAla Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser115 120 14 112 PRT Mus musculus 14 Glu Val Gln Leu Gln Glu Ser Gly ProAsp Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr ValThr Gly Tyr Pro Ile Thr Ser Gly 20 25 30 Tyr Thr Trp His Trp Ile Arg GlnPhe Pro Cys Leu Glu Trp Met Gly 35 40 45 Tyr Met His Tyr Asn Gly Ser ThrAsn Tyr Asn Pro Ser Leu Lys Ser 50 55 60 Arg Ile Ser Ile Thr Arg Asp ThrSer Lys Asn Gln Phe Phe Leu Gln 65 70 75 80 Leu Asn Ser Val Thr Thr GluAsp Thr Ala Thr Tyr Tyr Cys Glu Phe 85 90 95 Ser Ser Trp Asp Tyr Trp GlyGln Gly Thr Ser Val Thr Val Ser Ala 100 105 110

What is claimed is:
 1. A method for the detection of an analyte in abiological sample suspected of containing said analyte and aninterfering substance, said method comprising the steps of: a. forming atest mixture comprising i) said sample, ii) a binding partner specificfor said analyte, and iii) an interference reducing substance in aconcentration of at least about 10 μg/ml of the test mixture, saidsubstance comprising a peptide sequence derived from framework region 1or 3 of the variable domain of an antibody selected from the groupconsisting of said binding partner and antibodies used for immunologicaltherapy or scintigraphy, wherein said interference reducing substance isnot mouse serum and wherein said test mixture allows for binding of saidanalyte to said binding partner, and b. detecting said analyte presentin said sample by measuring the binding between said analyte and saidbinding partner.
 2. The method of claim 1 wherein said analyte is atumor marker.
 3. The method of claim 1 wherein said interferencereducing substance is an antibody or antibody fragment.
 4. The method ofclaim 1 wherein said peptide sequence is selected from the groupconsisting of SEQ ID Nos. 1-8 and partial sequences thereof, saidsequence having a length of at least 6 amino acids.
 5. The method ofclaim 1, wherein said analyte is CA125.
 6. The method of claim 1 whereinsaid antibody is selected from the group consisting of B72.3, and OC125.